Doctoral Program in Neurobiology and Behavior, Columbia University College of Physicians and Surgeons, New York, United States.
Department of Pharmacology, Columbia University College of Physicians and Surgeons, New York, United States.
Elife. 2017 Dec 19;6:e29744. doi: 10.7554/eLife.29744.
The functional repertoire of surface ion channels is sustained by dynamic processes of trafficking, sorting, and degradation. Dysregulation of these processes underlies diverse ion channelopathies including cardiac arrhythmias and cystic fibrosis. Ubiquitination powerfully regulates multiple steps in the channel lifecycle, yet basic mechanistic understanding is confounded by promiscuity among E3 ligase/substrate interactions and ubiquitin code complexity. Here we targeted the catalytic domain of E3 ligase, CHIP, to YFP-tagged KCNQ1 ± KCNE1 subunits with a GFP-nanobody to selectively manipulate this channel complex in heterologous cells and adult rat cardiomyocytes. Engineered CHIP enhanced KCNQ1 ubiquitination, eliminated KCNQ1 surface-density, and abolished reconstituted K currents without affecting protein expression. A chemo-genetic variation enabling chemical control of ubiquitination revealed KCNQ1 surface-density declined with a ~ 3.5 hr by impaired forward trafficking. The results illustrate utility of engineered E3 ligases to elucidate mechanisms underlying ubiquitin regulation of membrane proteins, and to achieve effective post-translational functional knockdown of ion channels.
表面离子通道的功能 repertoire 是通过运输、分拣和降解的动态过程来维持的。这些过程的失调是多种离子通道病的基础，包括心律失常和囊性纤维化。泛素化强有力地调节着通道生命周期中的多个步骤，但由于 E3 连接酶/底物相互作用的混杂性和泛素代码的复杂性，基本的机制理解仍然存在混淆。在这里，我们针对 E3 连接酶 CHIP 的催化结构域，使用 GFP 纳米抗体将其靶向 YFP 标记的 KCNQ1±KCNE1 亚基，以便在异源细胞和成年大鼠心肌细胞中选择性地操作这个通道复合物。工程化的 CHIP 增强了 KCNQ1 的泛素化，消除了 KCNQ1 的表面密度，并消除了重建的 K 电流，而不影响蛋白表达。一种能够实现泛素化化学控制的化学遗传变异，揭示了 KCNQ1 表面密度的下降是由于正向运输受损，大约 3.5 小时后才会下降。这些结果说明了工程化 E3 连接酶在阐明泛素调节膜蛋白的机制以及实现离子通道有效翻译后功能失活方面的应用。
